EP1293573A2 - Procédé pour la fabrication d'un vaccin marqueur contre un virus de mammifère - Google Patents

Procédé pour la fabrication d'un vaccin marqueur contre un virus de mammifère Download PDF

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Publication number
EP1293573A2
EP1293573A2 EP02018214A EP02018214A EP1293573A2 EP 1293573 A2 EP1293573 A2 EP 1293573A2 EP 02018214 A EP02018214 A EP 02018214A EP 02018214 A EP02018214 A EP 02018214A EP 1293573 A2 EP1293573 A2 EP 1293573A2
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Prior art keywords
virus
gene
plant
cereal
immunogenic
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EP02018214A
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German (de)
English (en)
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EP1293573A3 (fr
Inventor
Rainer Stahl
Birgit Dr. Nelsen-Salz
Norbert Dr. Wolf
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Maltagen Forschung GmbH
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Maltagen Forschung GmbH
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Publication of EP1293573A2 publication Critical patent/EP1293573A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a method for producing a marker vaccine against a mammalian virus and transgenic cereal plants or parts thereof, prepare the components of the vaccine.
  • KSP occurs not only in domestic pigs, but also in our domestic ones Wild boar on. This species has been reinforced again since the early 1990s affected by the plague. Wild boars infected with the KSP virus formed an important source of infection for ours in recent years House pig herds. 55% of KSP outbreaks in domestic pigs during the period 1993-1999 direct and indirect contacts with infected wild boar recycled. The control of the KSP in the wild boar population is therefore necessary to eliminate this source of infection for our domestic pig stocks.
  • the recombinant viral proteins are to be produced in the baculovector virus expression system (EP-A-0 924 298); After a pestivirus infection, animals develop antibodies (AK) against E rns , E2 (both coat proteins) and NS3 (a serine protease), of which only the AK against E2 have strong virus-neutralizing properties.
  • the marker vaccine After administration of the marker vaccine, the pig only forms AK against the coat protein contained in the vaccine and by using a specific diagnostic test it is then possible to distinguish between vaccinated and infected pigs.
  • the production and especially the application - each pig has to be vaccinated twice - are very time, labor and cost intensive. This vaccine is also not to be administered orally, so that it cannot be used in the wild boar population.
  • the present invention was based on the technical problem, another Way to produce marker vaccines, the disadvantages from the Does not have prior art. Another technical problem was that to provide funds required for the above-mentioned process.
  • the technical problems mentioned are solved by a A method of producing a marker vaccine against a mammalian virus, thereby is characterized in that the immunogenic protein or proteins of the vaccine from a cereal plant or parts thereof.
  • the further technical problem is caused by the provision of a transgenic cereal plant or parts thereof, containing at least one gene necessary for an immunogenic Encoded mammalian virus protein, resolved.
  • heterologous products especially heterologous polypeptides
  • transgenic plant material has become very important in recent years won.
  • certain Proteins are over- or under-expressed
  • transgenic plant material to produce heterologous products to construct that are not normally found in plants, e.g. B. bacterial Toxins, human proteins or bioplastics.
  • marker vaccine is understood to mean a vaccine that leads to an immunization in the immunized organism that results from the immunization distinguishes the organism by the actual pathogen. Usually generated the marker vaccine has a different pattern of antibodies than the actual pathogen. The immune response obtained can thus be used to distinguish whether an organism confronted with a marker vaccine or with the natural pathogen has been.
  • a "cereal plant” in the sense of the present invention is a plant which is in usually genetic due to a genetic modification of the genetic material Contains information necessary for at least one immunogenic protein - or at least immunogenic parts thereof - encoded which are part of the mammalian virus against which immunization in a mammalian organism is desired.
  • the immunogenic Protein is expressed by the cereal plant, possibly only after specific Induction of the promoter that regulates the expression of the immunogenic protein.
  • the immunogenic protein can optionally only be found in "parts" of the cereal plant be expressed. This will be the case regularly if the gene that encodes the immunogenic protein, under the control of a tissue-specific one Promoter stands. Accordingly, under “sharing" the cereal z. B. seeds, Understand leaves or tissue cultures of cereal plants.
  • a set of genes is expressed in the plant part of the immunogenic proteins (or rather immunogenic epitopes), but not all of the immunogenic proteins (epitopes) of the mammalian virus are encoded.
  • the marker vaccine can also be Modify addition of an immunogenic component so that the added component leads to an additional immune response that the natural pathogen does not causes.
  • a component can be, for example, a protein which is in does not occur in the natural pathogen, but is added to the marker vaccine and thus also in the treated organism to an antibody pattern leads from the antibody pattern generated by the natural pathogen different.
  • Such an additional protein or immunogenic epitope thereof can, for example, as a fusion partner with the actual immunogenic protein available.
  • the cereal plant barley, wheat, rye, triticale, rice or corn In particular are preferred cereal plants that are suitable for feeding animals such. B. wheat, barley, corn.
  • the marker vaccine is for immunization against a virus from the pestivirus family, in particular an immunization against the classical swine fever virus, the border Disease virus or the virus of bovine viral diarrhea occurs.
  • the immunogenic protein is selected from the gene product of the E2 gene or E ms gene of the classical swine fever virus.
  • Derivatives of the gene products mentioned can also be used, provided they can still cause adequate immunization against the virus mentioned.
  • the derivative can also be shortened forms of the complete protein, provided that at least some of the immunogenic epitopes are retained.
  • the derivative can also be a fusion protein of the E2 or E ms gene product with a further protein, the further protein serving as the actual "marker", as explained above.
  • the derivative can also be mutants of the protein product mentioned, which have been optimized, for example, in such a way that they produce an optimal immune response or the stability of the protein z. B. is improved in the cereal plant.
  • the codons of the Genes optimized to be optimal for the cereal plant in which the marker vaccine is located to be manufactured. This enables maximum expression of the desired protein in the cereal plant. The highest possible protein content is also in terms of immunization properties of that made from the plants Marker vaccine desirable.
  • the expression of the immunogenic takes place Proteins only in parts of the plant, such as the seeds, whereby in particular promoters are used that are either only during germination or grain filling are active.
  • Such expression systems are for example in US 5,693,506 and WO 97/32986.
  • This approach allows the immunogenic protein not to be in the field but only during germination in a closed production room in the plant or to synthesize the seed.
  • This so-called malting technology deals with with the germination of plant material under artificial conditions. Usually this technology is applied and embraced during the production of beer Steps such as soaking the plant material and subsequent germination controlled conditions. Under these "controlled” conditions, parameters such as moisture, temperature, salt concentrations and movement of the plant material controlled by human intervention.
  • Such processes are e.g. B. described in "The Beer Brewery” Volume 1, "The Technology of Malt Preparation” Needles Enke-Verlag Stuttgart (1999).
  • the present invention also relates to a transgenic cereal plant or the like Parts containing at least one gene for an immunogenic protein of a mammalian virus encoded, the gene being a so-called transgene.
  • Transgenic plant material is a plant material
  • the transgenic host cells contains, wherein the transgenic host cells in their genome at least one nucleic acid molecule have integrated stable, which is either heterologous in terms of the plant material or which is normally in untransformed cells is not available or which is homologous with regard to the plant material, but in the transgenic cells in a different genomic environment located as in the wild type cells.
  • the transgenic plant contains a set Genes that encode part of the immunogenic proteins of the mammalian virus.
  • one such a plant is based on the idea that the plant is not all immunogenic Epitopes of a pathogen are expressed, but only a subgroup that after immunization with the subset of epitopes, a different antibody pattern is obtained as after infection of the organism with the intact pathogen. Based on the different immune responses between immunized organisms and infected organisms, the immunized organisms from the infected can be distinguished.
  • the plant is selected from Barley, wheat, rye, triticale, rice and corn.
  • the plant preferably contains genes which code for proteins of a pestivirus.
  • the gene expresses in the transgenic plants encodes an immunogenic protein of a virus selected from the classical swine fever virus, the border disease virus and the virus of bovine viral diarrhea.
  • the genes of the superficial proteins of the named Viruses are known and are available to the person skilled in the art in the prior art.
  • the E2 gene and the E ms gene of the classical swine fever virus are particularly preferred for expression in the transgenic plant, the gene also being able to be modified, for example by targeted mutagenesis, in such a way that derivatives of the natural gene product were produced in the plant.
  • This can e.g. B. may be advantageous to positively influence the stability of the gene products or their immunogenic properties.
  • the desired gene product especially expressed in the cereal grain of the plant.
  • an appropriate Promoter e.g. activated under malting conditions, can be thus the desired gene product at any time by induction of the corresponding promoter.
  • the present invention further relates to animal feed or food that is a cereal plant or parts thereof, as described above. These compositions can then z. B. oral administration to stimulate the immune system against appropriate viruses.
  • Animal feed has decisive advantages over the techniques of hitherto practiced Vaccination by injection, because feeding animal feed is much easier is to be carried out and also z. B. for immunization of wild animals without further is possible.
  • the gene for a surface protein is used for the optimal yield of expression product of an animal pathogenic pathogen, subjected to a "codon optimization". Changes in the coding sequence of the gene become such made that the codons of the wild type by the optimal for the host plant Codons are replaced without the amino acid sequence of the encoded protein to change. In the examples, the optimal sequence for barley was determined.
  • the "chimeric gene” refers to a gene construct that contains a DNA sequence that is necessary for encodes a fusion protein being an (i) N-terminal unit of a signal sequence and one (ii) directly to the C-terminal region of the amino acid sequence protein portion of a mature heterologous following this signal sequence Contains protein.
  • the immunogenic structural proteins used for expression originate from pestiviruses, the genus pestivirus essentially consisting of the virus of classical swine fever (KSPV), the border disease virus (BDV) and the virus of the bovine viral diarrhea (BVDV).
  • the pestivirus genome consists of a (+) strand RNA of approximately 12.5 kb in length with a single open reading frame which codes for a polyprotein of approximately 4,000 amino acids.
  • the polyprotein is processed co- and post-translationally by cellular and viral proteases.
  • the viral structural proteins which include the envelope proteins E2, E rns and E1, are located in the N-terminal region of the polyprotein.
  • the native or synthetic gene can be modified with a view to an increased expression of the gene or stability of the expressed proteins in the plant tissue. For example, directed mutagenesis, exon shuffling and the like can be used to change the gene sequence.
  • the chimeric gene construct 5 'of the coding region comprises a promoter of a monocot plant.
  • Suitable promoters are the D-hordein promoter (SEQ ID NO: 3) and the amylase promoter (SEQ ID NO: 4) of the barley, which e.g. B. also in Whittier RF et al . (1987) Nucleotide sequence analysis of alpha-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells. Nucl. Acids Res . 15 , 2515-2535 and S ⁇ rensen MB et al. (1996) Hordein promoter methylation and transcriptional activity in wildtype and mutant barley endosperm. Mol . Gene. Genet . 250, 750-60 are described.
  • the chimeric gene preferably contains between the promoter and the coding sequence the 5'-untranslated region (5'-UTR) and 3 'of the coding sequence the non-translated region (3'-UTR) of a plant or bacterial gene ,
  • a preferred 3'-UTR is the nos terminator sequence.
  • This sequence comprises the non-coding sequence 5 'from the polyadenylation site, the polyadenylation site and the transcription termination sequence and is described, inter alia, in Jensen LG et al . (1998) Inheritance of a codon-optimized transgene expressing heat stable (1,3-1,4) -beta-glucanase in scutellum and aleurone of germinating barley. Hereditas 129 : 215-225.
  • the chimeric gene of the invention is described in an appropriate expression vector for plants was introduced.
  • the vector contains special elements of plasmids or viruses that allow DNA is transported into the plants by the bacteria.
  • Such expression vectors for Plants are e.g. described in US 5,693,506.
  • the vectors contain selection markers for use in plant cells, e.g. the nptll kanamycin resistance gene or the phosphinotricin acetytransferase gene, as well as sequences, which select and grow in secondary hosts such as Yeast or Bacteria, allow.
  • selection markers for use in plant cells, e.g. the nptll kanamycin resistance gene or the phosphinotricin acetytransferase gene, as well as sequences, which select and grow in secondary hosts such as Yeast or Bacteria, allow.
  • the vectors used for this purpose contain regions that form a Agrobacterium tumefaciens vector are homologous, here especially the T-DNA border sequences.
  • Embryos are prepared from immature seeds and after 48 hours of incubation co-cultivated with transformed agrobacteria. Different washing steps follow and incubation first in the dark, later in the light.
  • the expression rate of the foreign protein is first determined in the immature later determined in the ripe grain.
  • the animals are preferably immunized against domestic or wild boar a viral infection, preferably classic swine fever.
  • This includes the Transformation of plant material with a vector defined above, the cultivation and propagating the transformed plant, harvesting the desired plant material and the subsequent, preferably repeated feeding of the harvested material, e.g. Barley grains or malt to animals; that's it Material either untreated or malted.
  • the DNA of the vector encoded protein purified from the plant material and as a feed supplement administered to the animals.
  • the codons of the gene for the structural protein E2 of the KSP virus are optimized in such a way that the GC-rich triplets in question are used for the individual amino acids.
  • the gene is assembled from a total of 56 oligonucleotides (54 with a length of 40 nucleotides each and two with a length of 20 nucleotides): In the two oligonucleotides of the upper and lower strand, which contain the stop codon, is direct inserted a SacI restriction site after the stop codon for later cloning steps.
  • the oligonucleotides are assembled using ligation and PCR.
  • the codon-optimized gene is inserted by means of a "splice-PCR" (see, for example, Horton et al ., 1989, Gene 77 : 61-68) behind a 3 'fragment of the hordein promoter with a hordein signal peptide set that includes an Nco I interface in the front part.
  • this fragment (consisting of the rear part of the hordein promoter, the hordein signal peptide and the complete E2 gene) is cut out via Ncol and SacI restriction sites and cloned into a vector which is a complete hordein Promoter, a Hordein signal peptide and a Nos terminator.
  • a construct (RS265; FIG. 1) is obtained which consists of the hordein promoter, hordein signal peptide, E2 gene and Nos terminator in pUC18.
  • construct RS265 Two vectors suitable for the transformation of Agrobacterium of the Agll strain are now assembled.
  • One construct contains a so-called single cassette, the other a double cassette, which differs from the single cassette in that the two chimeric genes (1) ubiquitin promoter, marker gene, Nos terminator and (2) hordein promoter, hordein signal peptide , E2 gene, Nos terminator are each surrounded by a left and a right T-DNA border region in order to enable later independent integration into the plant genome.
  • the entire unit consisting of the ubiquitin promoter, marker gene, Nos terminator and hordein promoter, hordein signal peptide, E2 gene, Nos terminator is only bordered by a common T-DNA border region.
  • the construct RS265 described above is first cleaved with Eco RI, then the overhanging ends are filled in by a T4 DNA polymerase treatment and then a Hind III cleavage is carried out.
  • the purified fragment on which the E2 gene is located is ligated into a pUC18 vector which carries a unit consisting of the ubiquitin promoter, marker gene and Nos terminator.
  • This vector is cleaved with Bam HI and Hind III before ligation, and here too the Bam HI restriction site is filled in before Hind III cleavage.
  • the vector RS266 (FIG.
  • the fragment with the E2 gene is cut out of the construct RS265 described above by means of Hind III and Eco RI, again, as described above, the Eco RI restriction site being filled in before the Hind III cleavage.
  • the vector pAM300 (FIG. 5) specially designed for the creation of double cassettes already contains a left and right T-DNA border region, as well as a unit consisting of an ubiquitin promoter, marker gene and Nos terminator. This vector is cut with Not I / Hind III, the Not I interface being filled in here. The Hind III / Eco RI fragment from the RS265 is then cloned into this vector.
  • a unit consisting of ubiquitin promoter, marker gene, Nos terminator and Hordein promoter, Hordein signal peptide, E2 gene, Nos terminator is separated out by a left and right border sequence using Eco RI and Hind III and cut out accordingly sliced pRS2600.
  • RS269 FIG. 6
  • agrobacteria of the Agll strain are transformed by means of electroporation.
  • Barley plants - preferably of the Golden promise variety - are grown in climatic chambers, for example. After approx. 2-3 months, immature barley seeds, whose embryos have a diameter of approx. 1.5 mm - 2.5 mm, can be harvested.
  • the barley seeds are sterilized and prepared from the grains after washing and divided lengthways. Under certain circumstances it can make sense here divide the embryos before dissecting them by simply using the seed is shared. In this case, the divided embryo is dissected out after cutting. The embryos are incubated at 24 ° C for 48 h.
  • the barley embryos are washed several times until no more turbidity of the medium due to bacteria is visible. After moving The embryos on the selection plates are aerated Incubation room incubated in the dark for two weeks at 24 ° C.
  • the selection plates contain growth hormone and a selection agent, e.g. Bialaphos. To The embryos are transferred to new plates for 2 weeks and only sprouts and roots removed generously. The plates are again in two weeks Incubate in the dark at 24 ° C.
  • the embryos are transferred again and then kept under light that is especially suitable for plants. In this phase, the calli should develop green areas. If the plantlets are large enough, they are transferred to bowls to form roots.

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EP02018214A 2001-09-18 2002-08-20 Procédé pour la fabrication d'un vaccin marqueur contre un virus de mammifère Withdrawn EP1293573A3 (fr)

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Application Number Priority Date Filing Date Title
DE10145969A DE10145969A1 (de) 2001-09-18 2001-09-18 Verfahren zum Herstellen eines Markerimpfstoffs gegen ein Säugervirus
DE10145969 2001-09-18

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EP1293573A2 true EP1293573A2 (fr) 2003-03-19
EP1293573A3 EP1293573A3 (fr) 2003-12-17

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003013598A2 (fr) * 2001-08-09 2003-02-20 Lam Dominic M K Nouvelles compositions de vaccins et procedes de preparation de vaccins pour des maladies veterinaires et humaines
CN102711446A (zh) * 2009-11-06 2012-10-03 谷万达公司 表达细胞壁降解酶的植物以及表达载体
US10988788B2 (en) 2009-11-06 2021-04-27 Agrivida, Inc. Plants expressing cell wall degrading enzymes and expression vectors

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103805615A (zh) * 2014-02-19 2014-05-21 孙洁 密码子优化的非洲猪瘟病毒p54基因、其核酸疫苗及应用
US9993544B2 (en) * 2014-05-23 2018-06-12 Boehringer Ingelheim Vetmedica Gmbh Recombinant classical swine fever virus (CSFV) comprising substitution in the TAV epitope of the E2 protein

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WO1997010347A1 (fr) * 1995-09-15 1997-03-20 Howard John A Cassettes d'expression et procedes d'administration de vaccins veterinaires
WO2000037610A2 (fr) * 1998-12-23 2000-06-29 Boyce Thompson Institute For Plant Research At Cornell Expression des antigenes de surface de l'hepatite b immunogenes dans les plantes transgeniques

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US5693506A (en) * 1993-11-16 1997-12-02 The Regents Of The University Of California Process for protein production in plants

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1997010347A1 (fr) * 1995-09-15 1997-03-20 Howard John A Cassettes d'expression et procedes d'administration de vaccins veterinaires
WO2000037610A2 (fr) * 1998-12-23 2000-06-29 Boyce Thompson Institute For Plant Research At Cornell Expression des antigenes de surface de l'hepatite b immunogenes dans les plantes transgeniques

Non-Patent Citations (1)

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Title
DE SMIT A J: "Laboratory diagnosis, epizootiology, and efficacy of marker vaccines in classical swine fever: A review" VETERINARY QUARTERLY, Bd. 22, Nr. 4, Oktober 2000 (2000-10), Seiten 182-188, XP009019420 ISSN: 0165-2176 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003013598A2 (fr) * 2001-08-09 2003-02-20 Lam Dominic M K Nouvelles compositions de vaccins et procedes de preparation de vaccins pour des maladies veterinaires et humaines
WO2003013598A3 (fr) * 2001-08-09 2004-01-08 Dominic M K Lam Nouvelles compositions de vaccins et procedes de preparation de vaccins pour des maladies veterinaires et humaines
CN102711446A (zh) * 2009-11-06 2012-10-03 谷万达公司 表达细胞壁降解酶的植物以及表达载体
CN103966279A (zh) * 2009-11-06 2014-08-06 谷万达公司 一种处理植物生物量的方法
CN102711446B (zh) * 2009-11-06 2017-06-30 谷万达公司 表达细胞壁降解酶的植物以及表达载体
US10988788B2 (en) 2009-11-06 2021-04-27 Agrivida, Inc. Plants expressing cell wall degrading enzymes and expression vectors

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DE10145969A1 (de) 2003-04-10
US20030077640A1 (en) 2003-04-24
CN1422665A (zh) 2003-06-11

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